Mitochondria are major intracellular targets for oxygen-radical, and quinone toxicity. Much of the damage observed may be site-specific since mitochondria continuously generate oxygen-radicals during normal respiration, and redox-cycle a wide range of quinonoid compounds (cellular quinones, drugs, environmental toxins) to produce extremely high levels of oxygen radicals and other activated oxygen species. An example of this process is the severe cardiotoxicity produced by many of the anthracycline chemotherapeutic agents (eg adriamycin) in which mitochondrial enzymes and membranes are severely damaged, and which (at least in part) appears to be due to mitochondrial redox-cycling of the drug. The long-term goal of this research is to describe and define the overall mitochondrial toxicology, and tissue specificity, of oxygen-radicals and redox-cycling (cellular, pharmaceutical, and environmental) quinones. The objective of this application is to begin to test for causal relationships between mitochondrial production of oxygen radicals, and impairment of mitochondrial bioenergetic functions (electron transport, proton pumping, transmembrane potential, ATP synthesis, etc). Oxygen radicals (and other activated oxygen species) will be generated in two ways: 1) By adding agents (antimycin A, DCCD, FCCP, etc.) or using incubation conditions (high O2, substrates) which augment the native production of oxygen radicals by endogenous mitochondrial electron transport chain components (ubiquinone, flavins), and 2) by incubating mitochondria in the presence of oxidizable substrates and adding exogenous quinonoid compounds (menadione, adriamycin, daunorubicin, rubiazzone, p-bonzoquinone, and purified quinonoid compounds from exhaust fumes) which can redox-cycle with the electron transport chain, or with ancillary redox enzymes. Preliminary studies indicate that protein damage plays a large role in the loss of bioenergetic functions, and that damaged proteins are selectively degraded. Both damage and degradation must, therefore, be studied if an accurate profile of protein alterations is to be gained. In addition, mitochondrial lipid peroxidation also appears to be involved in some of the bioenergetic defects induced by oxygen radicals. The extent to which toxicity results from protein damage and protein degradation, rather than from lipid peroxidation, will be assessed using powerful definitive, and appropriate HPLC methods.